US10751448B2 - Multi-layer skin substitute products and methods of making and using the same - Google Patents

Multi-layer skin substitute products and methods of making and using the same Download PDF

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US10751448B2
US10751448B2 US15/542,095 US201615542095A US10751448B2 US 10751448 B2 US10751448 B2 US 10751448B2 US 201615542095 A US201615542095 A US 201615542095A US 10751448 B2 US10751448 B2 US 10751448B2
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layer
cells
live mammalian
hydrogel
mammalian
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US20170354763A1 (en
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Anthony Atala
Gayoung Jeong
James J. Yoo
Sang Jin Lee
Young-Joon Seol
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Wake Forest University Health Sciences
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Wake Forest University Health Sciences
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    • C12N2502/1305—Adipocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/80—Hyaluronan

Definitions

  • the present invention concerns live, artificial, skin substitute products and methods of making and using the same, such as for wound treatment and compound testing.
  • the current “gold standard” for skin replacement is the use of autologous skin grafts.
  • this treatment is often limited for patients.
  • most current engineered skins or skin substitutes do not fully recapitulate native skin as they are devoid of multiple skin cell types and structures like trilayers and dermal appendages.
  • the current commercially available skin cellular models are also limited as they only use either immortalized cell lines derived from skin tumors or one or two primary cell types (e.g., keratinocytes and/or dermal fibroblasts) to be simple; thus they do not well represent and replicate the complexity of in vivo skin.
  • an artificial mammalian skin substitute product comprising:
  • a first (“hypodermis-like”) layer comprising live mammalian adipocytes (e.g., induced pre-adipocytes) in a first hydrogel carrier;
  • a second (“dermis-like”) layer contacting or directly contacting the first layer and comprising live mammalian fibroblast cells and live mammalian follicle dermal papilla cells in combination in a second hydrogel carrier;
  • a third (“epidermis-like”) layer contacting or directly contacting the second layer (i.e., on the opposite side thereof as the first layer, so that the second layer is sandwiched between the first and third layers when the first layer is present), the third layer comprising live mammalian keratinocytes and live mammalian melanocytes in combination in a third hydrogel carrier.
  • first, second, and/or third hydrogel carriers comprise cross-linked hyaluronic acid, and/or the second and/or third hydrogel carriers optionally but preferably further comprise collagen.
  • the second and third layers are at least partially cross-linked with one another (either directly, or through an intervening cross-linkable layer).
  • the first layer is present and the first and second layers are at least partially cross-linked with one another.
  • each of the first layer when present, the second layer, and the third layer have overlying surface areas of from 0.5, 1 or 10 square centimeters up to 50, 200 or 400 square centimeters, or more.
  • the live mammalian adipocytes are human adipocytes; said live mammalian fibroblast cells are human fibroblast cells, said live mammalian follicle dermal papilla cells are human follicle dermal papilla cells, said live mammalian keratinocytes are human keratinocytes, and said live mammalian melanocytes are human melanocytes.
  • Some embodiments further comprise antigen-presenting dendritic cells or precursors thereof between the first layer and the second layer, in the second layer, between the second layer and the third layer, and/or in said third layer (e.g., in a total amount of from 1 or 2 million to 8 or 10 million (preferably 4 to 6 million) cells per cubic centimeter).
  • Some embodiments further comprise neural cells or precursors thereof between said first layer and said second layer, in said second layer, between said second layer and said third layer, and/or in said third layer (e.g., in a total amount of from 1 or 2 million to 8 or 10 million (preferably 4 to 6 million) cells per cubic centimeter).
  • FIG. 1 Effects of serum level on the cell viability (3D).
  • FIG. 5 Hair growth in bioprinted skin substitute construct after 3 weeks of growth in vitro.
  • FIG. 6 Data collection and 3D printing of Biomask mold using CAD/CAM modeling.
  • FIG. 7 Views of the porous polyurethane layer, the epidermis-like layer with keratinocytes, and the dermis-like layer with fibroblasts, of the formed Biomask.
  • FIG. 8 Schematic of printing each of the layers of the Biomask.
  • FIG. 9 Schematic design of Biomask structure and in vivo application onto a wound.
  • polyurethane mold is porous (300 ⁇ M pore size).
  • “Mammalian” as used herein refers to both human subjects (and cells sources) and non-human subjects (and cell sources or types), such as dog, cat, mouse, monkey, etc. (e.g., for veterinary or research purposes).
  • Hydrogel as used herein may be any suitable hydrogel.
  • the hydrogel includes water and is further comprised of or derived from polyalkylene oxides, poloxamines, celluloses, hydroxyalkylated celluloses, polypeptides, polysaccharides, carbohydrates, proteins, copolymers thereof, or combinations thereof, and more particularly are comprised of or derived from poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-polypropylene oxide) block copolymers, carboxymethyl cellulose, hydroxyethyl cellulose, methylhydroxypropyl cellulose, polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, alginate, gelatin, collagen, albumin, ovalbumin, copolymers thereof, and combinations thereof, all of which are
  • a cross-linked hyaluronic acid hydrogel (optionally including additional polymers such as gelatin) is preferred.
  • Antigen presenting dendritic cells including precursors thereof (e.g., induced or differentiated embryonic stem cells; CD34+ bone marrow precursor cells) are known. See, e.g., U.S. Pat. Nos. 6,008,004 and 8,785,189.
  • Neural stem cells including precursors thereof, are known. See, e.g., U.S. Pat. Nos. 6,001,654 and 8,785,187.
  • Vascular cells including precursors thereof and cells that can self-organize into a vascular network, are known. See, e.g., US Patent Application Publication Nos. US 20140273220 and US20140201988.
  • Products of the invention may be made by the steps of:
  • a first (“hypodermis-like”) layer comprising live mammalian adipocytes (e.g., induced pre-adipocytes) in a first hydrogel carrier on a substrate (e.g., an inert substrate such as a porous polymer mesh; collagen, etc.; or a wound on a subject in need of treatment); then (preferably within one half hour or less);
  • a substrate e.g., an inert substrate such as a porous polymer mesh; collagen, etc.; or a wound on a subject in need of treatment
  • the first hydrogel carrier when deposited, is deposited in prepolymerized or partially polymerized form; the second hydrogel carrier is deposited in prepolymerized or partially polymerized form; and/or the third hydrogel carrier is deposited in prepolymerized or partially polymerized form.
  • the depositing steps (a) and (b) are carried out under conditions in which said first hydrogel in said first layer, when present, and said second hydrogel in said second layer at least partially crosslink with one another; and said depositing steps (b) and (c) are carried out under conditions in which said second hydrogel in said second layer and said third hydrogel in said third layer at least partially crosslink with one another.
  • the layers may be crosslinked directly, or through an intervening cross-linkable layer.
  • first, second, and/or third hydrogel carriers comprise cross-linked hyaluronic acid, and/or the second and/or third hydrogel carriers optionally but preferably further comprise collagen.
  • the depositing is carried out under conditions in which the second and third layers are at least partially cross-linked with one another, and/or the first layer and second layers are at least partially cross-linked with one another—typically by carrying out the depositing steps sufficiently close in time so that cross-linking reaction between the two layers may occur.
  • intervening layer(s) can be interposed between the first and second hydrogen layers, and/or the second and third hydrogel layers, with the first and second, and/or second and third, hydrogel layers optionally cross-linked with their respective intervening hydrogel layer(s).
  • partial intervening layer is meant that the layer has openings therein through which the first and second, and/or second and third, layers directly contact one another.
  • additional cell types such as described below may optionally be deposited with such intervening layers.
  • the hydrogels of these intervening layer(s), when present, may be formed of the same materials as the first, second, and/or third hydrogel layers, and like those layers may be deposited in partially crosslinked form.
  • said first layer when present, has a thickness of from 100, 200 or 300 micrometers up to 400, 600 or 800 micrometers;
  • said second layer has a thickness of from 100, 200 or 300 micrometers up to 400, 600 or 800 micrometers;
  • said third layer has a thickness of from 100, 200 or 300 micrometers up to 400, 600 or 800 micrometers;
  • said product has a total thickness of from about 200, 400 or 600 micrometers up to 800, 1200 or 1600 micrometers when said first layer is absent, or a total thickness of 300, 600 or 900 micrometers up to 1200, 1800 or 2400 micrometers when said first layer is present.
  • each of the first layer when present, said second layer, and said third layer have overlying surface areas of from 0.5, 1 or 10 square centimeters up to 50, 200 or 400 square centimeters.
  • Cells may be included in any suitable amount.
  • said adipocytes are included in said first hydrogel carrier in an amount of from 1 or 2 million to 8, 10, 15 or 20 million (preferably 4 to 6 million or 10 to 20 million) cells per cubic centimeter; and/or (ii) said fibroblast cells and said dermal papilla cells included in said second hydrogel carrier in a ratio of about 8:1 or 6:1 to 2:1 or 1:1 (preferably 5:1 to 3:1) and/or at a combined density of about 5 or 8 million to 15, 20, 25 or 30 million (preferably about 10 million or about 20-25 million) cells per cubic centimeter; and/or (iii) said keratinocytes and said melanocytes included in said third hydrogel carrier in a ratio of about 20:1 or 10:1 to 8:1, 5:1, 3:1 or 2:1 (preferably from 12:1 to 3:1) and/or at a combined density of about 5 or 8 million to 15, 20, 25, 30 or 35 million (preferably about 10 million or about 20-30 million) cells per cubic
  • said live mammalian adipocytes are human adipocytes; said live mammalian fibroblast cells are human fibroblast cells, said live mammalian follicle dermal papilla cells are human follicle dermal papilla cells, said live mammalian keratinocytes are human keratinocytes, and/or said live mammalian melanocytes are human melanocytes.
  • the method may further comprise depositing antigen-presenting dendritic cells or precursors thereof between said first layer and said second layer, in said second layer, between said second layer and said third layer, and/or in said third layer (e.g., in a total amount of from 1 or 2 million to 8 or 10 million (preferably 4 to 6 million) cells per cubic centimeter).
  • the method may further comprise depositing neural cells or precursors thereof between said first layer and said second layer, in said second layer, between said second layer and said third layer, and/or in said third layer (e.g., in a total amount of from 1 or 2 million to 8 or 10 million (preferably 4 to 6 million) cells per cubic centimeter).
  • the method may further comprise depositing vascular cells or precursors thereof between said first layer and said second layer, in said second layer, between said second layer and said third layer, and/or in said third layer (e.g., in a total amount of from 1 or 2 million to 8 or 10 million (preferably 4 to 6 million) cells per cubic centimeter).
  • the construct has a diameter or width of from 1 to 5 millimeters, or from 3 to 7 millimeters, or from 5 to 10 millimeters, or from 8 to 16 millimeters, or from 10 to 20 millimeters, or from 20 to 50 millimeters, or from 30 to 80 millimeter, or from 50 to 100 millimeters.
  • Depositing can be carried out by any suitable technique, including but not limited to spraying, spreading/painting, coating, etc.
  • the depositing steps are carried out by printing or bioprinting in accordance with any suitable technique, including both “ink jet” type printing and syringe injection type printing.
  • Apparatus for carrying out such bioprinting is known and described in, for example, Boland et al., U.S. Pat. No. 7,051,654; Yoo et al., US Patent Application Pub. No. US 2009/0208466; and Kang et al., US Patent Application Publication No. US 2012/0089238.
  • the products described above When deposited on an inert substrate, the products described above may be removed therefrom and used immediately, or maintained and further propagated on that support in vitro in any suitable culture media.
  • the products may be packaged (with or without the support, or transferred to a different support) in a sterile container or package for subsequent use if desired, along with appropriate nutrients and/or culture media.
  • the support may be porous or non-porous.
  • the support may be a porous filter, membrane or mesh that is permeable to media nutrients for diffusion to the live cells of the construct, e.g., of one or more of the layers.
  • a wound such as a burn, incision (including surgical incision), abrasion, laceration or the like on a subject may be treated by topically applying a skin substitute product as described herein to that wound in a treatment-effective configuration (e.g., sufficiently covering or overlying the wound to aid in the healing thereof).
  • a treatment-effective configuration e.g., sufficiently covering or overlying the wound to aid in the healing thereof.
  • the first “hypodermis-like” layer may not be required.
  • Suitable subjects include both human subjects, and other animal (typically mammalian) subjects (e.g., dogs, cats, cows, pigs, sheep, horses, etc.) for veterinary (including veterinary medicine and pharmaceutical screening) purposes.
  • the wound may be a facial wound, such as a wound of the forehead, glabella, nasion, nose (e.g., nasal bridge, rhinion, infatip lobule, supratip, columella, alar-sidewall), nasolabial fold, philtrum, lips, chin, cheek, jaw, ear (e.g., helix, scapha, antihelical fold, antihelix, antitragus, lobule, tragus, concha, fossa), skin surrounding the eye (e.g., eyelid), etc.
  • a facial wound such as a wound of the forehead, glabella, nasion, nose (e.g., nasal bridge, rhinion, infatip lobule, supratip, columella, alar-sidewall), nasolabial fold, philtrum, lips, chin, cheek, jaw, ear (e.g., helix, scapha, antiheli
  • the live skin product may be fabricated on a customized mold made of an inert substrate in order to provide a personalized shape for wound healing.
  • the mold may be fabricated based on clinical image data such as CT data, optionally modified to impart the desired shape and features for the wound healing.
  • the mold may be formed from a polymeric material (e.g., polyurethane), optionally dispensed from a printer as taught herein.
  • the wound may be the result of a surgery or other medical procedure, such as plastic surgery.
  • an epidermis layer is deposited on the inert substrate, a dermis layer is deposited on the epidermis layer, and optionally a hypodermis layer is deposited on the dermis layer (depending on the nature of the wound and the need for the hypodermis in the wound treatment).
  • the live skin product comprising an inert substrate layer is molded to snugly fit onto the complex contour, shape and architecture of facial wounds.
  • one or more cell types of the product are autologous with respect to the subject to be treated. In some embodiments, one or more cell types of the product are allogenic with respect to the subject to be treated.
  • Live skin substitute products as described herein may be used as an alternative to live animal testing for compound or composition screening (e.g., screening for efficacy, toxicity, penetration, irritation, or other metabolic or physiological activity). Such testing may be carried out by providing a skin substitute product as described herein under conditions which maintain constituent cells of that product alive (e.g., in a culture media with oxygenation); applying a compound or composition to be tested (e.g., a drug candidate, typically provided in a vehicle or carrier, a topical composition such as a soap or cosmetic, etc.) to that product (e.g., by topical application to said third layer); and then detecting a physiological response (e.g., damage, scar tissue formation, irritation, penetration, cell proliferation, etc.) to said skin substitute product (e.g., burn, cell death, marker release such as histamine release, cytokine release, changes in gene expression, etc.), the presence of such a physiological response indicating said compound or composition has therapeutic efficacy, toxicity, irriation, pentration,
  • a control sample of the skin substituted may be maintained under like conditions, to which a control compound or composition (e.g., physiological saline, compound vehicle or carrier) may be applied, so that a comparative result is achieved, or damage can be determined based on comparison to historic data, or comparison to data obtained by application of dilute levels of the test compound or composition, etc.
  • a control compound or composition e.g., physiological saline, compound vehicle or carrier
  • the live skin substitute construct is form on and/or provided on an insert configured to be placed into a cell culture dish (e.g., a petri dish, a 2-well plate, a 6-well plate, a 12-well plate, a 24-well plate, 48-well plate, 96-well plate, etc.), such as a cell culture insert.
  • a cell culture dish e.g., a petri dish, a 2-well plate, a 6-well plate, a 12-well plate, a 24-well plate, 48-well plate, 96-well plate, etc.
  • Cell culture inserts are known and described in, e.g., U.S. Pat. Nos. 5,652,142, 5,578,492, 5,468,638, 5,470,473, etc.
  • the ratio between dermal fibroblasts and follicle dermal papilla cells was kept to be 4:1 and the cell density was also kept at 10 million cells/ml.
  • pre-adipocytes were pre-cultured and induced in monolayer for 2-3 days with pre-standardized differentiation media (Promocell, Germany) and induced adipocytes (iAd) were used to print at 5 million cells/ml.
  • Hydrogel Preparation Commercially available hyaluronic acid-gelatin based hydrogels (HyStem®-C, ESI-BIO, Alameda, Calif.) were used as the ‘biopaper.’ More specifically, 1% thiolated hyaluronic-acid (Glycosil)-1% thiolated gelatin (Gelin-S) with 2-4% PEGDA (Extralink) or 4-arm-PEGA (Creative PEGworks, Winston Salem, N.C., USA) were used as crosslinking agents for various gelation time and stiffness. Also, human collagen (10-20% (v/v), 3 mg/ml, ESI-BIO) solution was added for dermis and epidermis layers when making hydrogels.
  • 3D Printing A customized 3D bioprinter (A. Skardal et al., Bioprinted Amniotic Fluid-Derived Stem Cells Accelerate healing of Large Skin Wounds, Stem Cells Translational Medicine 1, 792-802 (2012)) was used. Cells and mixed cells and hydrogels ('bioink' and tiopaper') were loaded into a sterile syringe for each different layer to print. 300 ⁇ m sized nozzle and 20-80 kPa pressures were used depending on viscosity of each hydrogel, and cell/gel solution was printed according to an evenly-spaced, coil-shaped pattern at various scan speeds. In general, 6-8 constructs were printed per a syringe loading.
  • each layer was 300-350 ⁇ m.
  • the entire trilayered structures were about 900-1200 ⁇ m in thicknesses.
  • each skin construct printed inside the insert was placed in a 6-well plate, and was cultured with 5% heat inactivated serum keratinocyte based media (5% HKM, Promocell, Germany). For immersion culture, 300-400 ⁇ l and 2.5-3m1 of 5% HKM for inside and outside of the inserts were used, respectively, and media was changed every other day.
  • the bioprinted skin constructs were cultured in a standard cell culture incubator with constant temperature at 37° C. and 5% CO 2 .
  • FIG. 1 Serum free KGM2 media (Promocell) showed the lowest cell viability and 5% and 10% serum added KGM2 media seemed to be comparable to each other and most optimal in terms of cell viability qualitatively.
  • the qualitative results from Live/Dead were confirmed by two times of MTS cell viability assays: [1] five skin cells intermixed within hydrogels and [2] individual skin cell encapsulated within hydrogels.
  • Bioprinting of trilayered 3D in vitro skin constructs epidermis: keratinocytes+melanocytes; dermis: dermal fibroblasts+follicle dermal papilla cells; hypodermis: pre-adipocytes
  • epidermis keratinocytes+melanocytes
  • dermis dermal fibroblasts+follicle dermal papilla cells
  • hypodermis pre-adipocytes
  • FIG. 5 shows a bioprinted skin substitute construct similar to that of FIG. 4 , three weeks after topical implantation onto a mouse, with a hair follicle formed therein and a hair growing from that follicle.
  • the bioink used for printing consisted of 2 parts (Glycosil):2 parts (Gelin-S):1 part (8% (w/v) 4-Arm PEG-Acrylate, MW 5 k (PSB-423) from Creative PEGWorks and 10-20% (v/v) 3 mg/ml human collagen solution: VitroCol)).
  • the pressure varied from 40-80 kPa depending on the cell number or density of the Bioink.
  • the printed constructs were cultured in 5% fetal bovine serum containing keratinocyte growth medium for up to 3 weeks and further analyzed for cell distribution and viability.
  • Burn injury to the face remains one of the greatest challenges in wound care, and treatment may greatly affect the quality of life and social integration of the affected individuals.
  • the varied contours and complex movement of the face has been a challenge in repairing the facial wounds.
  • Current treatment strategies following injuries often lead to scarring, infection, graft failure and poor cosmetic outcome.
  • a customized engineered skin substitute that snuggly fits in to the complex contour, shape and architecture of facial wounds was developed using a 3-D fabricated Biomask ( FIG. 6 ) having a customized face-shaped structure combined with skin cells.
  • An integrated organ printing (IOP) system was used to fabricate the Biomask containing a face-shaped porous polyurethane (PU) with a layer containing human primary keratinocytes and a layer containing human primary dermal fibroblasts ( FIG. 7 ). Each component was precisely dispensed and placed by the control of air pressure and 3-axes stage.
  • IOP integrated organ printing
  • PU Polyurethane
  • High temperature around 150 degree-centigrade
  • high pressure (1500 kPa) were used for dispensing the PU.
  • keratinocytes and fibroblasts were mixed in hydrogel, respectively.
  • the PU structure supports the upper hydrogel structure of the epidermis and dermis layers. After implantation, the PU structure can act like a dressing for the skin wound.
  • the printed constructs were validated in a mouse full-thickness skin wound model.
  • the printed constructs were applied to the full-thickness skin wounds (1 ⁇ 1 cm 2 ) of nu/nu mice.
  • the engineered skin constructs were delivered with the porous PU layer. H&E-stained histological sections of skin samples, harvested at 14 days, showed clear differences in the quality of the epidermal layers near the center of the wound areas between control (nontreated) and engineered skin groups.
  • the Biomask provides the ability to deliver multiple cell types in a precise manner and maintain customized contours while facilitating rapid wound coverage and closure, which is critical in case of complex facial full-thickness wounds.
  • Clinical use of the Biomask fabricated with the 3-D TOP system is expected to enhance wound healing and skin regeneration and provide personalization to enhance functionality and cosmetic appearance of healed wounds.

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